Methods of tfeb activation and lysosomal biogenesis and compositions therefor

Abstract

The present disclosure pertains to methods of activating TFEB independent of mTORC1 activity, methods of activating TFEB by enhancing GABARAP/FNIP/FLCN complex localization at an intracellular membrane surface, methods of characterizing a TFEB activating agent, and methods of treating a TRPML1-associated disease, disorder or condition, and compositions for use in said methods.

1 . A method of activating TFEB independent of mTORC1 activity, the method comprising a step of:
 contacting a system that comprises:
 a membrane comprising LAMP-1, vATPase or GABARAP; and 
 components of a GABARAP/FLCN/FNIP complex; 
   with a TRPML1 agonist such that level of the GABARAP/FLCN/FNIP complex at the membrane is elevated.   
     
     
         2 . The method of  claim 1 , wherein the membrane comprising LAMP-1 vATPase or GABARAP defines a compartment. 
     
     
         3 . The method of  claim 2 , wherein the compartment is or comprises a lysosome. 
     
     
         4 . The method of  claim 1 , wherein the membrane is or comprises a lysosomal membrane. 
     
     
         5 . The method of  claim 5 , wherein the lysosomal membrane is part of an intact lysosome. 
     
     
         6 . The method of  claim 3  or  claim 6 , wherein the lysosome is in a cell. 
     
     
         7 . A method of activating TFEB independent of mTORC1 activity, the method comprising a step of:
 administering a TRPML1 agonist.   
     
     
         8 . The method of  claim 7 , wherein the step of administering comprises contacting a system with the TRPML1 agonist, wherein the system comprises:
 a lysosomal membrane; and   components of a GABARAP/FLCN/FNIP complex.   
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the system has a polymorphism or mutation in:
 a gene encoding a conjugation machinery protein (conjugation machinery gene) and/or   a gene encoding a component of the GABARAP/FLCN/FNIP complex.   
     
     
         10 . The method of  claim 9 , wherein the conjugation machinery gene is selected from the group consisting of Atg3, Atg5, Atg7, Atg12, Atg16L1, and combinations thereof. 
     
     
         11 . The method of  claim 10 , wherein the conjugation pathway gene is Atg16L1. 
     
     
         12 . The method of  claim 11 , wherein the polymorphism is T300A. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the TRPML1 agonist is of a chemical class selected from the group consisting of polypeptides, nucleic acids, lipids, carbohydrates, small molecules, metals, and combinations thereof. 
     
     
         14 . The method of  claim 8 , wherein the step of administering comprises exposing the system to the TRPML1 agonist under conditions and for a time sufficient that enhanced expression or activity of one or more CLEAR network genes and/or enhancement of one or more of detectable exocytosis activity, autophagy, clearance of lysosomal storage material, and lysosomal biogenesis is observed in the system relative to that prior to the exposure. 
     
     
         15 . The method of  claim 8 , wherein the step of administering comprises exposing the system to the TRPML1 agonist under conditions and for a time sufficient that enhanced expression or activity of one or more genes selected from Table 1 is observed in the system relative to that prior to the exposure. 
     
     
         16 . The method of  claim 7 , wherein the TRPML1 agonist is characterized in that, when assessed for impact on expression of CLEAR network genes, it shows a more restricted impact than that observed under starvation conditions. 
     
     
         17 . The method of  claim 1  or  claim 7 , wherein the TRPML1 agonist is characterized in that TRPML1 level or activity is higher in its presence than in its absence, under comparable conditions. 
     
     
         18 . The method of  claim 1  or  claim 7 , wherein the TRPML1 agonist is a direct agonist in that it interacts with TRPML1. 
     
     
         19 . The method of  claim 1  or  claim 7 , wherein the TRPML1 agonist is an indirect agonist in that it does not directly interact with TRPML1. 
     
     
         20 . A method of treating a TRPML1-associated disease, disorder or condition, the method comprising a step of:
 administering a TRPML1 agonist to a subject suffering from, or susceptible to, the TRPML1-associated disease, disorder or condition.   
     
     
         21 . The method of  claim 20 , wherein the TRPML1-associated disease, disorder or condition is or comprises an inflammatory condition. 
     
     
         22 . The method of  claim 20 , wherein the TRPML1-associated disease, disorder or condition is or comprises a lysosomal storage disorder. 
     
     
         23 . The method of  claim 20 , wherein the TRPML1-associated disease, disorder or condition is or comprises a polyglutamine disorder. 
     
     
         24 . The method of  claim 20 , wherein the TRPML1-associated disease, disorder or condition is or comprises a neurodegenerative proteinopathy. 
     
     
         25 . The method of  claim 20 , wherein the TRPML1-associated disease, disorder or condition is an infectious disease. 
     
     
         26 . The method of  claim 20 , wherein the TRPML1-associated disease, disorder or condition is selected from a group consisting of Crohn's Disease, Pompe Disease, Parkinson's Disease, Huntington's Disease, Alzheimer's Disease, Spinal-bulbar muscular atrophy, α-1-antitrypsin deficiency, and multiple sulfatase deficiency. 
     
     
         27 . The method of  claim 20 , wherein the TRPML1-associated disease, disorder or condition is Crohn's Disease. 
     
     
         28 . A method of activating TFEB by enhancing GABARAP/FNIP/FLCN complex localization at an intracellular membrane surface. 
     
     
         29 . The method of  claim 28 , wherein the intracellular membrane surface is a cytosolic surface of an intracellular compartment. 
     
     
         30 . The method of  claim 29 , wherein the intracellular compartment is a lysosome. 
     
     
         31 . The method of  claim 29 , wherein the intracellular compartment is a mitochondria. 
     
     
         32 . The method of  claim 29 , wherein the intracellular compartment is an endoplasmic reticulum. 
     
     
         33 . The method of any one of  claims 28 - 32 , wherein the method comprises administering a TRPML1 agonist. 
     
     
         34 . The method of any one of  claims 28 - 33 , wherein TFEB activation is independent of mTORC1 activity. 
     
     
         35 . A method of characterizing a TFEB activating agent, the method comprising:
 assessing effect on FLCN localization and/or level of a GABARAP/FNIP/FLCN complex at one or more intracellular membrane surfaces.   
     
     
         36 . A method of treating a conjugation-machinery-associated (“CMA”) disease, disorder or condition or a GABARAP/FNIP/FLCN complex-associated disease, disorder or condition, the method comprising a step of:
 administering a TRPML1 agonist. 
 
     
     
         37 . The method of  claim 27 , wherein the disease, disorder or condition is or comprises Crohn's Disease. 
     
     
         38 . A method comprising a cellular assay for characterizing activators of TFEB, TFE3 and/or MITF, wherein the cellular assay comprises cells comprising:
 (a) presence of a vATPase small molecule inhibitor;   (b) genetic disruption of ATG8 conjugation machinery;   (c) presence of a small molecule inhibitor of ATG8 conjugation machinery;   (d) genetic disruption of a member of a GABARAP subfamily of proteins;   (e) mutation of a LIR domain in FNIP1 or FNIP2;   or a combination thereof.   
     
     
         39 . The method of  claim 38 , wherein the vATPase small molecule inhibitor is Bafilomycin A1. 
     
     
         40 . The method of  claim 38 , wherein the vATPase small molecule inhibitor is not an analogue of Salicylihalamide A. 
     
     
         41 . The method of  claim 38 , wherein the genetic disruption of ATG8 conjugation machinery comprises knock-out of a gene, knock-in of a gene, expression of one or more mutant alleles, siRNA, shRNA, antisense, or a combination thereof. 
     
     
         42 . The method of  claim 38 , wherein the genetic disruption of the member of a GABARAP subfamily of proteins comprises knock-out of a gene, knock-in of a gene, expression of one or more mutant alleles, siRNA, shRNA, antisense, or a combination thereof.